靛玉红衍生物PHⅡ-7对人乳腺癌细胞株MCF-7的杀伤效应及其机制

The Cytotoxicity of Indirubin Derivative PHⅡ-7 against Human Breast Cancer MCF-7 Cells and Its Mechanisms

目的观察靛玉红衍生物PHⅡ-7对人乳腺癌癌细胞株MCF-7的杀伤作用并探讨其初步机制。方法四唑蓝(MTT)比色法检测细胞的增殖活性;Annexin V/PI双染法检测细胞凋亡率;PI染色结合流式细胞术检测细胞周期分布;DCFH-DA染色检测细胞内活性氧(reactive oxygen species,ROS)生成变化;RT-PCR法、Western blot法检测原癌基因c-fos基因及蛋白表达水平。结果不同浓度的PHⅡ-7对MCF-7细胞的增殖抑制率为43.13%～90.90%,抑制效应随着浓度增加而增强(P<0.05);各实验浓度的PHⅡ-7均能诱导细胞凋亡,1.25、2.5、5.0μmol/L PHⅡ-7作用24h后,MCF-7细胞的早期凋亡率分别为(1.43±0.02)%,(9.14±0.36)%,(45.79±8.46)%,具有浓度依赖性;PHⅡ-7处理MCF-7后致其G0/G1期和S期细胞比例下降,G2/M期细胞比例明显上升;PHⅡ-7作用于MCF-7细胞2h后,细胞内ROS水平显著增高;PHⅡ-7处理MCF-7后,原癌基因c-fos mRNA和蛋白表达呈浓度依赖性下调。结论 PHⅡ-7对MCF-7具有明显的体外杀伤作用,其作用机制可能与细胞周期阻滞、改变细胞氧化还原平衡状态及下调原癌基因表达有关。

Objective To observe the cytotoxicity of indirubin derivative PHⅡ-7 against human breast cancer MCF-7 cells and to study its primary mechanisms. Methods The proliferation of MCF-7 cells was detected using MTT colorimetry. Annexin Ⅴ/PI double staining was applied to detect the apoptosis rate of MCF-7 cells. The distribution of cell cycles was detected using PI staining and flow cytometry （FCM）. The levels of reactive oxygen species （ROS） in MCF-7 cells were detected by DCFH-DA staining. The mRNA and protein levels of c-fos were detected using RT-PCR and Western blot analysis. Results PHⅡ-7 at different concentrations inhibited the proliferation of MCF-7 cells in a concentration-dependent manner, with the inhibitory rate ranging from 43.13% to 90.90% （P0.05）. The inhibition was strengthened along with increased concentrations. PHⅡ-7 at different concentrations could induce the apoptosis of MCF-7 cells. The early apoptosis rate was 1.43% ± 0.02%, 9.14% ± 0.36%, and 45.79% ± 8.46%, respectively with the action of 1.25, 2.50, and 5.00 μmol/L PHⅡ-7, respectively, showing dose-dependent manner. FCM analysis found that the proportion of MCF-7 cells in the G0/G1 phase and the S phase decreased after treatment with PHⅡ-7, and the ratio of MCF-7 cells in the G2/M phase obviously increased （P0.01）. The intra-cellular ROS level was significantly elevated 2 h after pretreatment with PHⅡ-7. The levels of the protooncogene c-fos mRNA and protein were down-regulated in a dose-dependent manner after action of PHⅡ-7. Conclusions PHⅡ-7 exerted obvious in vitro cytotoxic effects on MCF-7 cells. Its mechanisms might be associated with arresting the cell cycle, regulating the redox equilibrium, and down-regulating the expression of the protooncogene.